Outer retinal thickness and visibility of the choriocapillaris in four distinct retinal regions imaged with spectral domain optical coherence tomography in dogs and cats.

PURPOSE: To evaluate the outer retinal band thickness and choriocapillaris (CC) visibility in four distinct retinal regions in dogs and cats imaged with spectral domain optical coherence tomography (SD-OCT). To attempt delineation of a fovea-like region in canine and feline SD-OCT scans, aided by the identification of outer retinal thickness differences between retinal regions. METHODS: Spectralis® HRA + OCT SD-OCT scans from healthy, anesthetized dogs (n = 10) and cats (n = 12) were analyzed. Scanlines on which the CC was identifiable were counted and CC visibility was scored. Outer nuclear layer (ONL) thickness and the distances from external limiting membrane (ELM) to retinal pigment epithelium/Bruch's membrane complex (RPE/BM) and ELM to CC were measured in the area centralis (AC), a visually identified fovea-like region, and in regions superior and inferior to the optic nerve head (ONH). Measurements were analyzed using a multilevel regression. RESULTS: The CC was visible in over 90% of scanlines from dogs and cats. The ONL was consistently thinnest in the fovea-like region. The outer retina (ELM-RPE and ELM-CC) was thickest within the AC compared with superior and inferior to the ONH in dogs and cats (p < .001 for all comparisons). CONCLUSIONS: The CC appears a valid, albeit less than ideal outer retinal boundary marker in tapetal species. The AC can be objectively differentiated from the surrounding retina on SD-OCT images of dogs and cats; a fovea-like region was identified in dogs and its presence was suggested in cats. These findings allow targeted imaging and image evaluation of these regions of retinal specialization.

Development and validation of methods to visualize conventional aqueous outflow pathways in canine primary angle closure glaucoma.

PURPOSE: Angle closure glaucoma (PACG) is highly prevalent in dogs and is often refractory to medical therapy. We hypothesized that pathology affecting the post-trabecular conventional aqueous outflow pathway contributes to persistent intraocular pressure (IOP) elevation in dogs with PACG. The goal of this study was to determine the potential for aqueous angiography (AA) and optical coherence tomography (OCT) to identify abnormalities in post-trabecular aqueous outflow pathways in canine PACG. METHODS: AA and anterior segment OCT (Spectralis HRA + OCT) were performed ex vivo in 19 enucleated canine eyes (10 normal eyes and 9 irreversibly blind eyes from canine patients enucleated for management of refractory PACG). Eyes were cannulated and maintained at physiologic IOP (10-20 mmHg) prior to intracameral infusion of fluorescent tracer. OCT scleral line scans were acquired in regions of high and low perilimbal AA signal. Eyes were then perfusion fixed and cryosections prepared from 10/10 normal and 7/9 PACG eyes and immunolabeled for a vascular endothelial marker. RESULTS: Normal canine eyes showed segmental, circumferential limbal AA signal, whereas PACG eyes showed minimal or no AA signal. AA signal correlated with scleral lumens on OCT in normal dogs, but lumens were generally absent or flattened in PACG eyes. Collapsed vascular profiles were identified in tissue sections from PACG eyes, including those in which no lumens were identified on AA and OCT. CONCLUSIONS: In canine eyes with PACG, distal aqueous outflow channels are not identifiable by AA, despite normalization of their IOP, and intra-scleral vascular profiles are collapsed on OCT and histopathology.

Mapping retinal ganglion cell somas in a large-eyed glaucoma model.

Purpose: The purpose of this study was to identify a robust, representative region of interest (ROI) for studies of retinal ganglion cell (RGC) soma loss in feline congenital glaucoma (FCG), a spontaneous, large-eyed glaucoma model. Methods: Seven FCG and three wild-type (wt) eyes were collected from 10 adult cats of both sexes. Eyes enucleated postmortem were immediately fixed overnight in 4% paraformaldehyde and then stored in 0.1 M PBS at 4 °C. The retinas were wholemounted, Nissl stained with cresyl violet, and imaged using light microscopy. Somas of RGCs were manually identified according to long-established morphological criteria and quantified using a semiautomated method; their coordinates were used to create density maps and plots of the retinal topography. The RGC axon counts for the corresponding eyes were obtained from glutaraldehyde-fixed, resin-embedded optic nerve cross-sections stained with 0.1% p-phenylenediamine (PPD) using a semiautomated counting method. Correlations between total optic nerve axons and RGC soma counts were assessed by linear regression. A k-means cluster algorithm was used to identify a retinal ROI, with further definition using a probability density algorithm. Results: Interindividual variability in RGC total soma counts was more pronounced in FCG cats (mean = 83,244, range: 0-155,074) than in wt cats (mean = 117,045, range: 97,373-132,972). In general, RGC soma counts were lower in FCG cats than they were in wt cats. RGC axon counts in the optic nerve cross-sections were lower than, but strongly correlated to, the total RGC soma count across all cats (in wt and FCG retinas; R2 = 0.88) and solely FCG eyes (R2 = 0.92). The k-means cluster algorithm indicated a region of the greatest mean difference between the normal wt retinas and FCG-affected retinas within the temporal retina, incorporating the region of the area centralis. Conclusions: As in other species, RGC soma count and topography are heterogeneous between individual cats, but we identified an ROI in the temporal retina for future studies of RGC soma loss or preservation in a large-eyed model of congenital glaucoma. Many of the methods refined and established to facilitate studies in this FCG model will be broadly applicable to studies in other large-eyed models.

Microstructure and resident cell-types of the feline optic nerve head resemble that of humans.

The lamina cribrosa (LC) region of the optic nerve head (ONH) is considered a primary site for glaucomatous damage. In humans, biology of this region reflects complex interactions between retinal ganglion cell (RGC) axons and other resident ONH cell-types including astrocytes, lamina cribrosa cells, microglia and oligodendrocytes, as well as ONH microvasculature and collagenous LC beams. However, species differences in the microanatomy of this region could profoundly impact efforts to model glaucoma pathobiology in a research setting. In this study, we characterized resident cell-types, ECM composition and ultrastructure in relation to microanatomy of the ONH in adult domestic cats (Felis catus). Longitudinal and transverse cryosections of ONH tissues were immunolabeled with astrocyte, microglia/macrophage, oligodendrocyte, LC cell and vascular endothelial cell markers. Collagen fiber structure of the LC was visualized by second harmonic generation (SHG) with multiphoton microscopy. Fibrous astrocytes form glial fibrillary acidic protein (GFAP)-positive glial columns in the pre-laminar region, and cover the collagenous plates of the LC region in lamellae oriented perpendicular to the axons. GFAP-negative and alpha-smooth muscle actin-positive LC cells were identified in the feline ONH. IBA-1 positive immune cells and von Willebrand factor-positive blood vessel endothelial cells are also identifiable throughout the feline ONH. As in humans, myelination commences with a population of oligodendrocytes in the retro-laminar region of the feline ONH. Transmission electron microscopy confirmed the presence of capillaries and LC cells that extend thin processes in the core of the collagenous LC beams. In conclusion, the feline ONH closely recapitulates the complexity of the ONH of humans and non-human primates, with diverse ONH cell-types and a robust collagenous LC, within the beams of which, LC cells and capillaries reside. Thus, studies in a feline inherited glaucoma model have the potential to play a key role in enhancing our understanding of ONH cellular and molecular processes in glaucomatous optic neuropathy.

Relationship between corneal sensitivity, corneal thickness, corneal diameter, and intraocular pressure in normal cats and cats with congenital glaucoma.

OBJECTIVE: To determine the effect of feline congenital glaucoma (FCG) on corneal sensitivity, and relationships between corneal sensitivity, central corneal thickness (CT), and corneal diameter (CD). ANIMALS AND PROCEDURES: Corneal sensitivity (estimated by corneal touch threshold [CTT] using Cochet-Bonnet esthesiometry); CT using ultrasonic pachymetry; intraocular pressure (IOP) using rebound tonometry; and maximal horizontal CD were measured in 16 normal and 14 FCG cats, both males and females, aged 7 months-3.5 years. All procedures complied with an Institutional Animal Care and Use Committee-approved protocol. Data were analyzed by linear regression: paired Student's t tests for between-eye comparisons, and unpaired Student's t tests for comparisons between groups. Relationships between parameters were evaluated by Pearson correlation coefficients and linear mixed effects modeling. For statistical tests, with the exception of values that were Benjamini-Hochberg adjusted for multiple comparisons, P-values < 0.05 were considered significant. RESULTS: Mean CTT and CT values were lower in FCG eyes relative to normal eyes, but differences were not statistically significant. Mean CD was significantly larger in FCG eyes relative to normal eyes, and there was a significant negative correlation between CD and CTT in FCG (r = -0.8564, corrected P = 0.005). These associations were confirmed in linear mixed effects models. CONCLUSIONS: Eyes with FCG have significantly larger CDs when compared with normal eyes, and larger CDs correlated with decreased corneal sensitivity in this group. Further studies are warranted to explore the effect of buphthalmos and corneal enlargement on corneal sensitivity and innervation in feline subjects with chronic glaucoma.

Aqueous Humor TGF-ß2 and Its Association With Intraocular Pressure in a Naturally Occurring Large Animal Model of Glaucoma.

Purpose: Transforming growth factor (TGF)-ß2 has been widely implicated in human glaucoma pathology. The purpose of this study was to determine the source of TGF-ß2 in aqueous humor (AH) and its relationship with intraocular pressure (IOP) in an inherited large animal model of glaucoma. Methods: Sixty-six glaucomatous cats homozygous for LTBP2 mutation, and 42 normal cats were studied. IOP was measured weekly by rebound tonometry. AH was collected by anterior chamber paracentesis from each eye under general anesthesia, and serum samples collected from venous blood concurrently. Concentrations of total, active and latent TGF-ß2 in AH and serum samples were measured by quantitative sandwich immunoassay. For comparisons between groups, unpaired t-test or Mann Whitney test were used, with P < 0.05 considered significant. The relationships between TGF-ß2 concentrations and IOP values were examined by Pearson's correlation coefficient and generalized estimating equation. Results: IOP and AH TGF-ß2 concentrations were significantly higher in glaucomatous than in normal cats. AH TGF-ß2 showed a significant, robust positive correlation with IOP in glaucomatous cats (r = 0.83, R2 = 0.70, P < 0.0001). Serum TGF-ß2 did not correlate with AH TGF-ß2 and was not significantly different between groups. TGF-ß2 mRNA and protein expression were significantly increased in local ocular tissues in glaucomatous cats. Conclusions: Enhanced, local ocular production of TGF-ß2 with a robust positive association with IOP was identified in this spontaneous feline glaucoma model, providing a foundation for preclinical testing of novel therapeutics to limit disease-associated AH TGF-ß2 elevation and signaling in glaucoma.

Sub-region-Specific Optic Nerve Head Glial Activation in Glaucoma.

Glaucoma, a multifactorial neurodegenerative disease characterized by progressive loss of retinal ganglion cells and their axons in the optic nerve, is a leading cause of irreversible vision loss. Intraocular pressure (IOP) is a risk factor for axonal damage, which initially occurs at the optic nerve head (ONH). Complex cellular and molecular mechanisms involved in the pathogenesis of glaucomatous optic neuropathy remain unclear. Here we define early molecular events in the ONH in an inherited large animal glaucoma model in which ONH structure resembles that of humans. Gene expression profiling of ONH tissues from rigorously phenotyped feline subjects with early-stage glaucoma and precisely age-matched controls was performed by RNA-sequencing (RNA-seq) analysis and complementary bioinformatic approaches applied to identify molecular processes and pathways of interest. Immunolabeling supported RNA-seq findings while providing cell-, region-, and disease stage-specific context in the ONH in situ. Transcriptomic evidence for cell proliferation and immune/inflammatory responses is identifiable in early glaucoma, soon after IOP elevation and prior to morphologically detectable axon loss, in this large animal model. In particular, proliferation of microglia and oligodendrocyte precursor cells is a prominent feature of early-stage, but not chronic, glaucoma. ONH microgliosis is a consistent hallmark in both early and chronic stages of glaucoma. Molecular pathways and cell type-specific responses strongly implicate toll-like receptor and NF-κB signaling in early glaucoma pathophysiology. The current study provides critical insights into molecular pathways, highly dependent on cell type and sub-region in the ONH even prior to irreversible axon degeneration in glaucoma.

Imaging Distal Aqueous Outflow Pathways in a Spontaneous Model of Congenital Glaucoma.

PURPOSE: To validate the use of aqueous angiography (AA) in characterizing distal aqueous outflow pathways in normal and glaucomatous cats. METHODS: Ex vivo AA and optical coherence tomography (OCT) were performed in nine adult cat eyes (5 feline congenital glaucoma [FCG] and 4 normal), following intracameral infusion of 2.5% fluorescein and/or 0.4% indocyanine green (ICG) at physiologic intraocular pressure (IOP). Scleral OCT line scans were acquired in areas of high- and low-angiographic signal. Tissues dissected in regions of high- and low-AA signal, were sectioned and hematoxylin and eosin (H&E)-stained or immunolabeled (IF) for vascular endothelial and perivascular cell markers. Outflow vessel numbers and locations were compared between groups by Student's t-test. RESULTS: AA yielded circumferential, high-quality images of distal aqueous outflow pathways in normal and FCG eyes. No AA signal or scleral lumens were appreciated in one buphthalmic FCG eye, though collapsed vascular profiles were identified on IF. The remaining eight of nine eyes all showed segmental AA signal, distinguished by differences in time of signal onset. AA signal always corresponded with lumens seen on OCT. Numbers of intrascleral vessels were not significantly different between groups, but scleral vessels were significantly more posteriorly located relative to the limbus in FCG. CONCLUSIONS: A capacity for distal aqueous humor outflow was confirmed by AA in FCG eyes ex vivo but with significant posterior displacement of intrascleral vessels relative to the limbus in FCG compared with normal eyes. TRANSLATIONAL RELEVANCE: This report provides histopathologic correlates of advanced diagnostic imaging findings in a spontaneous model of congenital glaucoma.